Stronger antioxidant enzyme immunoreactivity of Populus tomentiglandulosa extract than ascorbic acid in rat liver and kidney

Objective(s): Populus species have various pharmacological properties, including antioxidant activity. In this study, the effects of Populus tomentiglandulosa extract (PTE) on histopathology and antioxidant enzymes in the rat liver and kidney were examined. Materials and Methods: Sprague-Dawley rats were assigned to three groups; (1) normal diet fed group, (2) ascorbic acid-containing diet-fed group as a positive control, (3) PTE-containing diet-fed group. The histopathology in the rat liver and kidney was examined by hematoxylin and eosin staining. The effect of PTE was examined in the rat liver and kidney by immunohistochemistry for antioxidant enzymes, such as superoxide dismutases (SOD1 and SOD2), catalase (CAT), and glutathione peroxidase (GPx). Results: No marked histopathological alterations were observed in the liver and kidney of the PTE-containing diet-fed group. In the liver, the mean numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells were significantly increased in the PTE-containing diet-fed rats, compared with those in the normal- and ascorbic acid-containing diet-fed rats. In the kidney, all SOD1, SOD2, CAT, and GPx immunoreactive structures were significantly increased in the PTE-containing diet-fed group, compared with those in the normal- and ascorbic acid-containing diet-fed groups. Conclusion: Results showed that PTE treatment significantly increased antioxidant enzymes in the rat liver and kidney, and we suggest that PTE might have hepato- and nephro-protective potentials against oxidative stress.


Introduction
Oxidative stress occurs due to an imbalance between the production and removal of reactive oxygen species (ROS), such as hydrogen peroxide (H 2 O 2 ), superoxide anions (O 2 •-), and hydroxy radicals ( • OH). The overproduction of ROS leads to the oxidation of cellular components and cell death (1,2). It has been reported that hepatopathy and nephropathy, which are results of hepatic and renal parenchymal injuries, are closely associated with the overproduction of ROS (3,4). Many researchers have reported endogenous antioxidant defense mechanisms including antioxidant enzymes (superoxide dismutase [SODs], catalase [CAT], and glutathione peroxidase [GPx]) to reduce and protect against oxidative stress (5)(6)(7). For example, SODs catalyze O 2 •into H 2 O 2 , and CAT and GPx convert H 2 O 2 and H 2 O, which plays coordination in the protection of oxidative damage (8). Therefore, it has been widely accepted that excessive oxidative stress is responsible for the onset and progression of cell damage/death in organs, including the liver and kidney (6,9,10).
The genus Populus (poplar) belongs to the Salicaceae family, and it has been well known that phenolic compounds and flavonoids are main components of poplar extracts, which are related to various pharmacological activities (11). In addition, many studies have reported that Populus species, such as Populus nigra, Populus alba, and Populus davidiana, display several pharmacological activities including antioxidant, antiinflammatory, and hepato-protective (12)(13)(14)(15)(16)(17).
Although pharmacological properties of some populous species have been well reported, few studies on pharmacological activities of Populus tomentiglandulosa (Korea poplar) have been demonstrated; only our recent study has shown that P. tomentiglandulosa extract (PTE) has a neuroprotective effect against transient cerebral ischemia-induced hippocampal neuronal damage by attenuation of reactive gliosis (18). In addition, no studies regarding the effects of PTE on normal visceral organs have been performed yet. In the present study, therefore, we examined whether treatment with PTE represents hepatotoxicity and nephrotoxicity in normal rats, and we investigated effects of PTE on SODs (SOD1 and SOD2), CAT, and GPx, in livers and kidneys of rats.

Preparation of PTE
P. tomentiglandulosa (specimen no.: KWNA200004212082, Kangwon National University) was collected in September 2016 in Kangwon province (South Korea) and kept in a deep freezer (-70 °C ). For the preparation of ethanol PTE, as described in our previous study (15), the stem and root bark of P. tomentiglandulosa were washed with distilled water, air-dried at 60 ± 0.5 °C and ground into a fine powder by using a grinder (IKA M20, IKA, Staufen, Germany). The powder was refluxed with 70% ethanol for 24 hr at 70 ± 0.5 °C . The extraction procedure was repeated three times, and the extract was filtered using Whatman No. 1 filter paper, concentrated in a vacuum evaporator and dried in a freeze-drier. The extraction yield was 14.5%. The percentage yield of the sample was calculated by comparing the weight of obtained dry extract to the initial weight of the sample.

Experimental animals and treatment of PTE
Male Sprague-Dawley rats (aged 12 weeks; body weight 300-320 g) were obtained from the Experimental Animal Center, Kangwon National University, Chuncheon, Republic of Korea. The rats were housed in a conventional state under adequate temperature (about 23 °C ) and humidity (60%) control with a 12-hr light/12-hr dark cycle. The rats were allowed free access to food and water. The procedures for animal handling and care were approved (approval no. KW-180124-1) by the Institutional Animal Care and Use Committee at Kangwon National University.
The animals were divided into 3 groups: (1) normalgroup (n=7), which served as a negative control group and received a normal composition pellets diet; (2) ascorbic acid-containing diet-fed group (AA-group) (n= 7), as a positive control group; and (3) PTE-containing diet-fed group (PTE-group) (n=7). As described in our previous studies (19,20), AA and PTE were mixed by 0.5% of pellets weight (w/w), respectively (Table 1). Each group was designated to receive different amounts composition of the pellets for 28 days. The age of the rats and administration period of PTE were selected based on our previous studies that showed antioxidant activities of some extracts derived from plants in the rat liver and kidney (19,20).

Tissue processing for histology
For histological examination, rats in each group (n=7) were anesthetized with sodium pentobarbital (60 mg/ kg, IP) (JW Pharmaceutical, Seoul, Korea) and perfused transcardially with 0.1 M phosphate-buffered saline (PBS) (pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate-buffer (PB) (pH 7.4) at 28 days after AA or PTE diet. Their livers and kidneys were removed and postfixed in 10% buffered formalin. The livers and kidneys were cut sagittally, embedded with paraffin, and sectioned into 6 μm thickness on a microtome (Leica, Wetzlar, Germany).

Hematoxylin and eosin (H-E) staining
To examine pathological changes in the livers and kidneys of each group, H&E staining was done according to the general protocol. The sections were stained with H&E solution, dehydrated, and mounted.

Immunohistochemistry for antioxidant enzymes
Immunohistochemistry was carried out for SOD1, SOD2, CAT, and GPx to examine changes in antioxidants in the livers and kidneys according to our published method (19,20). In brief, the sections were incubated with goat anti-SOD1 (1:500, Calbiochem, Darmstadt, Germany), goat anti-SOD2 (1:1000, Calbiochem), rabbit anti-CAT (1:1000, LabFrontier, Seoul, Korea), or sheep anti-GPx (1:1000, Chemicon International, Billerica, MA) as primary antibodies. Each negative control test was done by using a pre-immune serum instead of each primary antibody to establish the specificity of  Table 1. Composition of experimental diets for the study of antioxidant effects of Populus tomentiglandulosa extract in the liver and kidney each immunostaining. The negative controls showed no immunoreactivity in the sections of the livers and kidneys. For quantitatively analyzing SOD1, SOD2, CAT, and GPx immunoreactivity in the liver, digital images of the liver were taken around the central vein by using an AxioM1 light microscope (Carl Zeiss, Germany), which was equipped with a digital camera (Axiocam, Carl Zeiss, Germany) connected to a PC monitor. SOD1, SOD2, CAT, and GPx immunoreactive cells were counted at 200X magnification. Cell count in each group was obtained by averaging all numbers counted from each rat.
Ten sections of the kidney per animal were selected to quantitatively analyze SOD1, SOD2, CAT, and GPx immunoreactivity. Digital images of the kidney were captured at the renal cortex. The density of each SOD1, SOD2, CAT, and GPx-immunoreactive structure was evaluated based on optical density (OD): OD was obtained after the transformation of the mean gray level by using the formula (OD = log [256 /mean gray level]). The ratio of the OD of each immunoreactive image was calibrated as % (relative optical density, ROD) by using Adobe Photoshop version 8.0 and analyzed by using NIH Image 1.59 software (Adobe systems inc., San Jose, CA). The ratio of each ROD was calibrated as %, with the normal-group designated as 100 %.

Statistical analysis
All data in this study are shown as mean±SEM. A multiple-sample comparison was applied in order to test the differences between groups by ANOVA and the Tukey multiple range post hoc test using the criterion of the least significant differences. Statistical significance was considered at P<0.05.

H&E staining
Effects of PTE administration on the liver and kidney were evaluated using H&E staining (Figure 1). It was found that there were no marked histopathological alterations in the livers and kidneys of AA-and PTEgroups compared with those in the normal-group (Figure 1). These results indicate that AA and PTE did not affect normal hepatic and renal histology.

Discussion
In this study, no obvious histopathological changes were shown in the livers and kidneys of the AA-and PTE-groups, compared with the normal-group. These results indicate that PTE treatment might not lead to hepatotoxicity as well as nephrotoxicity.
To the best of our knowledge, there is no information on antioxidant effects and compounds from P. tomentiglandulosa, but some previous studies have reported antioxidant activities of other Populous species. For example, Debbache-Benaida et al. (12) reported that ethanolic extract from P. nigra buds displayed antioxidant potential and attenuated hepatotoxicity induced by aluminum exposure in mice. They also reported that phenolic acids were major components of the antioxidant activity of P. nigra, and they urged that the hepato-protective effect of P. nigra buds ethanolic extract could be due to radical scavenging activity (12). Dudonne et al. (13) reported that treatment with P. nigra bud extract significantly increased catalase gene expression in both normal and aged fibroblasts from facial skin. They suggested that the potential effect of P. nigra bud extract on catalase gene expression reduced the detrimental effect of oxidative stress, which was related to skin aging (13). In addition, Si et al. (21) reported that phenolic glycosides from P. ussuriensis Kom. exhibited excellent antioxidant activity, suggesting that P. ussuriensis could be used as a good antioxidant (21).
In the present study, we examined the effect of PTE on antioxidant enzymes in the rat liver and kidney, and we found that numbers of SOD1, SOD2, CAT, and GPx immunoreactive cells were significantly increased in the liver and immunoreactivities of the 4 antioxidant enzymes were significantly increased in the kidneys of the PTE-group compared to those in the AA-group as well as the normal-group. Many researchers have reported that increased levels and activities of antioxidant enzymes, including SODs, CAT, and GPx following treatment with plant-derived extracts and their compounds display beneficial effects in livers and kidneys. In this regard, researchers (22) showed that treatment with extract from Ocimum basilicum leaves minimized damage induced by acetaminophen in livers and kidneys of BALB/c mice, which was closely related to the decrease of malonaldehyde (MDA, an indicator of oxidative stress) level and increase of SOD and CAT levels (22). In addition, a study (23) demonstrated that treatment with extract and active compound (6-gingerol) from Zingiber officinale attenuated mercuric chloride-induced hepatorenal toxicity via reducing MDA level and restoring SODs, CAT, and GPx activities in rat liver and kidney (23). Interestingly, another study (10) also reported that treatment with poplar type propolis resulted in the improvement of streptozotocin-induced liver and kidney lesions, showing that hepatorenal GPx level was increased and MDA level was decreased (10). Based on the findings, they suggested that poplar type propolis could reduce hepatorenal injury by decreased lipid peroxidation and increase of anti-oxidant enzyme   (10). Therefore, it is likely that pretreatment with PTE might represent hepato-and nephroprotective effects via increasing antioxidant activity against hepatic and renal injury induced by oxidative stress.

Conclusion
The data show that PTE treatment significantly increased expressions of antioxidant enzymes in the rat liver and kidney. It is suggested that PTE might have hepato-and nephro-protective potentials against oxidative stress.